The present invention relates to a method, notably to a method for diagnosing the presence of a bacterium in a sample.
It is common practice to propagate bacteria from a sample of a bodily fluid, such as sputum, on a suitable culture medium so as to be able to detect the colonies of bacteria on the culture medium either visually or using a test reagent or assay. Such a technique is applicable over a wide range of bacteria and has become widely accepted as a standard bacteria detection and diagnostic procedure. However, problems arise where the bacterium is a slow growing type where it may take several days or even weeks before adequate growth of the bacteria has taken place to be detectable. This is particularly the case with some Mycobacterium or Legionella species, where it can take up to four weeks or more to propagate detectable colonies of the bacteria. This enforces an unacceptable delay in confirming infection with the bacteria in patients and it would be desirable to provide a more rapid assessment of the presence and identity of the bacteria.
It has been proposed in PCT Application No WO92/02533 to prepare a sample of a viable bacterium and to infect that with a strain of a virus which is specific to that bacterium, such bacteria-infecting virus being denoted as bacteriophage or, more commonly, phage. Some of the phage particles infect the bacteria and replicate within the bacteria cell. Those phage particles which are not absorbed by the bacteria remain with the mother liquor or matrix of the carrier of the bacteria, typically a culture medium. If the infected sample is then treated, for example with heat and/or an acid to kill, or is washed with a surfactant to remove, the exposed, exogenous, phage particles, the exogenous phage particles are inactivated or removed. The infecting endogenous, phage particles are not inactivated, since they are protected within the bacterial cell. The treated infected bacteria can then be cultured in the presence of further un-infected bacteria which are permissive to, that is can be infected by, the phage particles. The phage particles within the infected bacteria replicate causing the cell wall to rupture or lyse and release its load of replicated phage particles. This first generation of progeny phage particles infects other bacterial cells, causing successive cycles of infection replication and lysis. This gives an exponential increase in the number of phage particles in the culture medium. Either the reduced growth of the bacterial cells or the large number of replicated phase particles can be measured or observed, even where the initial number of infected bacterial cells was small, thus enhancing the sensitivity of the detection of the presence of the initial bacterial cells in the sample being assessed.
This modification enhances the sensitivity of detection of bacterial cells and has been used successfully in the detection of fast growing bacteria such as Salmonella spp. However, with slow growing bacteria such as Mycobacterium spp, we nave found that the time taken for this procedure to give a detectable population of phage particles remains substantially the same as with other techniques and the procedure does not solve the time problem with such bacteria. The need still exists for a procedure which achieves rapid and accurate detection of slow growing bacteria.
We have devised a method by which slow growing bacteria can be rapidly detected, thus overcoming the problems in having to wait days or even weeks for such detection. The method can also be used to determine the sensitivity of given bacteria to antibiotic and other drugs and to determine whether a given antibiotic or drug is active against given bacteria or whether a given virucidal composition is effective against a given virus. Since the method can be performed using simple apparatus and by unskilled persons, the method readily lends itself to operation in third world countries where skilled microbiologists and complex apparatus may not be available.
Accordingly, from one aspect the present invention provides a method for detecting the presence of first bacterium in a sample, which method comprises the steps of:
a. infecting a viable sample of the first bacterium cells with a phage which is specific to that bacterium whereby at least some of the bacterium cells each absorb one or more of the phage particles;
b. inactivating or removing the phage particles which have not been absorbed by the bacterium cells;
c. cultivating the infected first bacterium cells in a medium containing at least one other second bacterium which has a reproduction rate greater than the reproduction rate of the said first bacterium whereby at least some of the said second bacterium cells become infected by phage particles released upon the lysis of said infected first bacterium cells, each of said infected bacterium cells acting as a source for further infection of a subsequent generation of the infection cycle; and
d. monitoring either the population of replicated phage particles and/or the reduced growth of the said second bacterium in stepc to determine the presence of said first bacterium cells in the sample under test.
Preferably, the cultivation and infection of the second bacterium is carried out to or near the steady state at which further bacterial cell growth and lysis substantially ceases. At this state, a substantially steady population of dead bacteria cells due to the activity of the phage particles is present in the cultivation medium. The areas of the cultivation medium in which phage replication has taken place can be detected visually as clear spots in the surrounding cultivation medium which is more cloudy, opaque or otherwise visually different due to the growth of viable bacteria. These areas of reduced bacterial growth due to phage activity are commonly termed plaques. Since the second bacterium reproduces at a greater rate than the first bacterium, the time taken for the initial infected first bacterium cells to produce detectable plaques is greatly reduced. This acceleration in the development of plaques does not occur when the same bacterium is used for the initial phage infection and for the subsequent cultivation steps.
Alternatively, the presence of replicated phage particles in the cultivation medium at the end of stepc can be assessed using conventional phage assay techniques. This can be done in addition to or as an alternative to the determination of the existence of plaques as described above.
The method of the invention can thus be applied to the detection of slow crowing bacteria in a sample, for example from a patient suspected of being infected with Mycobacterium tuberculosis or Legionnaires disease, where a conventional assessment would takes days or weeks to give any observable indication that such bacteria were present in the initial sample. In such a case, a proportion or the initial sample is infected with a phase specific to the expected bacterium and the phage particles are carried through the in-activation step b because they are protected by the bacterial cells which they have infected. These protected phage particles cause the development of phage or plaques in the environs of each infected first bacterium cell in the cultivation of the infected cellsxe2x80x94stepc. If none of the suspected bacteria were present in the sample, substantially no phage or plaques would be formed in stepc and the cultivation medium would present a substantially uniform appearance.
The invention may also be carried cut using a known bacterium which has been treated with an antibiotic or other drug under test to determine whether the antibiotic or drug has any bactericidal effect. In this case, the population of phage or plaques at the end of stepc indicates the efficacy or otherwise of the antibiotic or drug in killing the initial bacterium. In a conventional test method, the antibiotic or drug would be applied to the bacterium and the number of dead cells would be a direct indication of the efficacy of the antibiotic or drug. However, it may be difficult to observe the individual dead cells except under a microscope. In this application of the invention, the treatment of the first bacterium with the antibiotic or drug, which may kill the first bacterium, is combined with infection of the remaining viable first bacterium with the phage particles to demonstrate how many of the first bacteria have escaped being killed by the antibiotic or drug.
In another application of the invention, a sample of a virus, or phage, is treated with a composition which has a virucidal or virus growth regulant effect. The treated phage is then used to infect a viable first bacterium which is permissive to that specific phage, that is the bacterium can be infected by that phage. The infected bacterium is then treated to in-activate the exogenous phage particles, that is those which do not infect the bacterium cells and remain exposed outside, or exogenous to, the cells. The in-activated infected first bacterium cell material is then cultured in the presence of a second, faster reproducing, bacterium to produce plaques where the phage particles in the infected first cells have replicated and lysed. If the initial treatment of the phage particles has been fully effective, no viable phage particles will be present when the first bacterium material is infected and, therefore, no viable phage particles will carry through to the cultivation in the presence of the second bacterium. Therefore, the second bacterium will reproduce to give a substantially uniform colour or opacity to the culture medium. If, however, the initial treatment of the phage particles was not fully effective, phage particles will be carried through to the cultivation of the second bacterium and will cause plaques, which can be detected visually.
For convenience, the invention will be described hereinafter in terms of the assessment of the presence of a slow growing Mycobacterium spp. The invention can be applied to bacteria from a wide range of aminal or vegetable hosts, but is of especial application in the detection of bacteria from mammal hosts and for convenience the invention will be described with especial reference to the assessment of such a slow growing bacterium in an initial sample of a bodily material from a human suspected of being infected with that bacterium.
The term slow growing bacterium is used herein to denote those bacteria which, under normal conditions of propagation at 37xc2x0 C. in a culture medium comprising 90% v/v of the medium known as 7H9 and 10% v/v of the nutrient known as OADC, have an effective reproduction cycle as assessed by a doubling of the bacterial cell population (the effective doubling rate) in excess of 10 hours. Such bacteria would normally take in excess of 72 hours using a conventional cultivation technique to produce detectable phage population or plaques, or are diffiult to cultivate. Using the method of the invention, phage or plaques can usually be detected within 16 hours with such bacteria.
Typical slow growing bacteria include Mycobacterium spp, notably Mycobacterium tuberculosis, Mycobacterium paratuberculosis and Mycobacterium avium; Legionella spp, notably Legionella pneumophila; Helicobacter spp, notably Helicobacter pylori; Streptococcus spp, notably Streptococcus adjacens; and Rikettsia spp, notably Rickettsia prowazekii. The invention may also be applied to bacteria which, whilst having an instantaneous doubling rate of less than 10 hours, have an average effective doubling rate greater than this due to the difficulty with which they initially propagate, for example Streptococcus defectens and Extremophiles spp. The term slow growing bacteria is therefore used herein and in the claims to denote bacteria which are inherently slow propagators and bacteria which have a slow average propagation rate over a period of ten hours due to intermittent failure or interruptions in their propagation cycle.
Whilst the invention is of especial benefit in detecting such slow growing bacteria, the invention can be applied to any bacterium where it is desired to increase the speed of formation of a detectable phage population or plaques, for example in the detection of bacteria which have a doubling rate shorter than 10 hours, for example in the range 4 to 10 hours, in human samples, food products or in samples taken to determine bacterial infestations in kitchens and the like, so as to provide more rapid testing for bacteria than would be achieved using conventional bacterial cultivation techniques. For convenience, the invention will be described hereinafter in terms of the use of Mycobacterium tuberculosis as the first bacterium.
The first bacterium can be present in any suitable form of sample from the patient, eg. sputum or other mucal secretions. However, the invention can be applied to samples of other bodily materials, for example, blood, urine or stools, from humans, other mammals, such as horses, cattle, sheep and pigs, and samples from vegetable matter, for example leaf or root crops or fruit. For convenience, the invention will be described hereinafter in terms of testing a sample of sputum from a human patient.
Typically, the sample will exist as a freshly drawn fluid in which the bacterium is still viable. However, where the sample has been frozen or dried for storage and/or transport prior to testing, it will usually be desired to hold the sample in a suitable culture medium at about 20 to 55xc2x0 C., preferably 37xc2x0 C., to ensure that the bacteria are viable and capable of infection with phage particles in step b of the method of the invention. If desired, the initial sample can be treated, for example thinned with N-acetyl cysteine and/or treated with alkali, surfactant or an acid, to reduce the population of other bacteria which might interfere with the detection of the bacterium under assessment. Such pre-treatments can be carried out using conditions and techniques commonly used in the preparation of bacterial samples for assessment. However, the need to decontaminate the initial sample from other bacteria is reduced as compared with conventional methods for assessing slow growing bacteria, since the method of the invention is more rapid than conventional test methods and the effect of the other bacteria is less marked.
The initial sample contains the bacterium to be assessed in a viable state. This will usually be in a fluid or gel, eg. agar, cultivation medium. However, the carrier medium could be a particulate solid or the pores of a foraminous material, such as a ceramic frit or a foamed plastic or a filter paper or the mesh apertures in a reticulate material, impregnated with a suitable nutrient composition. For convenience, the invention will be described hereinafter in terms of the use of a fluid carrier medium for the initial bacterium sample which is subsequently applied to a gel carrier medium.
The initial sample is infected with a suitable phage which is specific to the type of first bacterium under assessment and to the second bacterium in which the infected first bacterium is to be cultivated in stepc of the method of the invention. The phage may be generically specific to the type of bacterium, so that the phage will infect a range of bacteria within a genus. However, it may be preferred to use a narrowly specific phage where a specific bacterium is to be assessed. If desired, the phage may be modified to increase the specificity of the phage, so as to reduce infection of other bacteria in the sample being assessed. Suitable phage modification can be achieved by known techniques, for example by genetic modification, having regard to the specific bacterium to be infected, for example as described in PCT Application No WO92/02933. Typical phage for present use include AG1, B1, BG1, BK1, C3, D29, D34, D56A, GS4E, HP1, L1, I3 and TM4. The optimum phage for present use will depend upon the first and second bacterium used, since the phage or its replicand produced upon lysis from a bacterial cell needs to be permissive to the bacteria used. Thus, where the presence of Mycobacterium tuberculosis is being assessed using Mycobacterium smegmatis as the second bacterium, the phage D29 is suitable; and where Mycobacterium avium, is used, the phase TM4 is suitable.
The phage can be provided in the form of, for example, a cultivar in a fluid medium or can be provided in the form of freeze or spray dried particles. The phage is admixed with the viable bacterial sample using any appropriate technique and under any suitable conditions. Typically, the bacterial sample will be in the form of a fluid and the phage infection is applied in a fluid carrier to the bacterial sample.
It is preferred to allow the phage infection to proceed at temperatures of up to 55xc2x0 C., notably at about 37xc2x0 C., for from 10 minutes to 4 hours to allow infection of the majority of the bacterial cells with at least one phage particle each. However, excessive infection may so weaken the bacteria that they do not survive the in-activation of the non-absorbed phage particles using acid as described below. The optimum infection time for a given bacterium can be determined by simple trial and error tests or by microscopic examination of the infected bacteria, having regard to the conditions to be experienced in the subsequent stages of the method of the invention.
The infected bacterial sample is then treated to remove or in-activate any exogenous phage particles which have not been absorbed into the bacterial cells and which remain outside the bacterial cells and exposed to the conditions introduced during the in-activation stagexe2x80x94step b . For convenience, the term in-activation will be used herein to denote collectively any process which renders the non-absorbed phage particles substantially incapable of replication in the next step of the method of the invention. The term thus includes the physical separation or removal of the phage particles from the sample or the de-activation of the particles. This in-activation can be carried out using any suitable technique, for example those described in PCT Application No WO92/0233. Preferred techniques include washing and filtration of the infected sample to separate the exogenous phage particles physically from the infected bacterial cells; by treating the infected sample with an acid, surfactant or chemical to kill or render inactive the exogenous phage particles; or by preferentially absorbing the exogenous phage particles from the infected sample on a suitable substrate which can then be separated from the bacterial cells.
For convenience, the invention will be described hereinafter in terms of chemical or acid treatment of the mixture of infected bacterial cells and exogenous phage particles. Such treatment is preferably carried out using a mineral acid or a iron salt thereof, for example ferrous ammonium sulphate, or phosphoric or sulphuric acid; a C1-6 aliphatic carboxylic acid, notably acetic acid, which nay be diluted glacial acetic acid or vinegar; or an acidic buffer medium. The treatment achieves a pH value in the bacterial carrier medium which in-activates the phage used, but which does not kill the infected first bacterium. The optimum washing and pH conditions will depend upon the bacterium under assessment and can be determined by simple trial and error tests. Typically, the infected bacterial material is acidified and incubated at an elevated temperature, typically about 37xc2x0 C., for sufficient time, typically about 15 minutes, and the pH is then adjusted to about neutrality by the addition of an alkali or base.
In an alternative technique, the exogenous phage is killed by the addition of a virucide to the infected bacterial material. The optimal virucide will depend upon the phage to be killed and the nature of the first bacterium and can readily be determined by simple trial and error tests. Thus, suitable virucides for present use where the first bacterium is Mycobacterium tuberculosis include iron salts of mineral acids, and unsaturated fatty acids and alcohols containing from 12 to 24 carbon atoms, optionally with actinic or other radiation, notably UV radiation at about 400 to 600 nm wave length.
After in-activation, the bacterial material contains viable first bacterial cells having inside at least some of them viable phage particles. This mixture is mixed with a cultivation mixture containing a second bacterium or mixture of bacteria. These second bacteria are characterised in that they are infectable by the phage particles within the cells of the first bacteria, that is they are permissive to the phage; and in that they propagate at a rate which is greater than the propagation rate of the first bacterium under assessment. It is preferred that the second bacterium has a doubling rate which is at least twice the doubling rate of the first bacterium. The optimal relative rate of propagation will depend upon the actual rate of propagation of the first bacterium, in that a high relative rate of propagation will be recurred with a very slow growing first bacterium if the time taken to produce a detectable phage population and/or reduced second bacterial growth areas or plaques s not to be excessive. Typically, the second bacterium will have a doubling rate of from 4 to 20 times that of the first bacterium. Thus, for the assessment of Mycobacterium tuberculosis in the initial sample, we have found that the use of Mycobacterium smegmatis as the second bacterium gives particularly rapid results. Other suitable second bacteria for present use include other Mycobacterium, for example Mycobacterium aurum. 
If desired, the infected first bacterium can be allowed to stand after the in-activation step b to achieve at least one generation of replication and release of the phage particles from the infected cells, before admixture with the second bacterium. Such standing allows the replicated phage particles to be released from the infected bacterial cells so as to provide more sites of potential infection within the second bacterial cells which assists the detection of low numbers of viable first bacterium in the initial sample.
In stepc of the method of the invention, the infected first bacterium is cultivated in the presence of the second, faster propagating bacterium. When the phage particles replicate within the infected first bacterium cells, they form a larger number of phage particles which are released from the infected cells as the cells lyse. Each released phage particle can infect an adjacent bacterial cell, which may be a first or second bacterium cell. Each of the phage particles in those newly infected bacterial cells can replicate and are released to infect further bacteria cells; and so on.
Cultivation of the mixture of first and second bacteria can be carried out using any appropriate techniques and conditions. Typically, the second bacteria will be used in the form of a cultivation in a fluid or gel medium which is mixed with the infected first bacteria. However, the second bacteria may be used in the form of a freeze or spray dried powder or as solid particles having the bacteria impregnated or coated thereon. For convenience, the invention will be described hereinafter in terms of the application of the second bacterium in an agar gel carrier to the infected first bacterium. The amount of the second bacterium added to the treated first bacterium sample can vary over wide ranges depending upon the expected amount of first bacterium cells in the sample and the desired theme within which a result from the method of the invention is required. In general, it is preferred to provide a greater number of second bacterium cells, typically from 2 to 50 times, than the number of first bacterium cells so as to enhance the probability that a phage particle lysed from an infected first bacterium cell will infect a second bacterium cell rather than another first bacterium cell. The optimum ratio of first to second bacterium cells can readily be established by simple trial and error tests
During the cultivation process, it is preferred that the cultivation medium change in some detectable way to reflect the presence of replicated phage particles, viable second bacteria or plagues of dead bacterial cells due to the action of the phage particles. Typically, growth of the second bacteria will cause cloudiness or colour in an agar cultivation medium and the phage particles and associated dead bacterial cells from which they have been released will result in less cloudy or clear spots, plaques, within the coloured or cloudy medium, so that the plaques can be detected and assessed visually. Alternatively, the areas of differential growth can be detected by differential staining using conventional staining techniques. Where the cultivation medium contains appropriate ingredients, the bacterial cells can release lumiphores when they lyse and these can be detected under UV radiation, for example as described in PCT Application WO92/02633. The increased numbers of replicated phage particles can also be detected by immunoassay techniques, for example ELISA, or another enzymatic technique.
The number of phage particles and/or of plaques gives an indication of the number of first bacterium cells present and hence the presence or otherwise of such cells in the initial sample to be assessed.
As indicated above, the method of the invention provides a rapid test for the presence of an expected bacterial strain in the initial sample from the patient. By suitable selection of the phage particles used to infect the first bacteria, the test can be made specific to a particular bacterium. Alternatively, the test can provide a broad indication as to the type of bacteria present in the initial sample. The method of the invention can thus be tailored to the results required. Since the results can be obtained in a matter of hours rather than days as hitherto required, it is practicable to carry out an initial general screen, followed by a specific test once the general type of bacterium present in the initial sample has been identified. Since the method of the invention can be carried out using simple cell culture techniques and the results can be assessed visually, the method of the invention can readily be used in third world countries where skilled labour and complex laboratory facilities may not be available.
The invention also provides a diagnostic test kit for use in the method of the invention to detect the presence or absence of a first bacterium in a sample, which kit comprises a source of a known phage infection for the first bacterium; a source of a viable second bacterium which has a doubling rate which is faster than that of the first bacterium and which is permissive to the phage particles to be released from the first bacterium infected by said phage infection. Preferably also, the kit comprises means for in-activating and/or removing the exogenous phage particles from first bacteria infected by said source of phage infection.